Method for producing fragrant alcohols

ABSTRACT

This invention relates generally to methods and compositions for producing a sesquiterpene alcohol comprising contacting a sesquiterpene with a P450 polypeptide with monooxygenase activity.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/023,640 filed Mar. 21, 2016, which is a national stage applicationunder 35 U.S.C. § 371 of International Patent ApplicationPCT/EP2014/070060 filed on Sep. 19, 2014, which claims the benefit ofU.S. provisional application 61/880,149, filed on Sep. 19, 2013. Theentire contents of each of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 9000US_DIV_SequenceListing. The size of the textfile is 423 KB, and the text file was created on Jan. 16, 2018.

FIELD

The field relates to cytochrome P450s and uses to produce sesquiterpenealcohols.

BACKGROUND

Terpenes hydrocarbons such as alpha and beta santalenes have beenproduced via biochemical processes for example such as throughgenetically altered cells. These terpenes and the alcohol derived fromthem are major constituents of sandalwood oil and the alcohols areimportant perfumery ingredients typically obtained commercially throughthe distillation of the heartwood of Santalum species (e.g.,Sandalwood). Examples of such alcohols include α-sinensol, β-sinensol,α-santalol, β-santalol, α-trans-bergamotol and epi-β-santalol. Althoughnew biochemical pathways have been developed, including geneticallyengineered cells, to generate the terpene hydrocarbons, it is desirableto find a biochemical pathway to generate and produce the alcoholsderived from the santalenes. It is further desirable to use abiochemical pathway to not only generate such alcohols but it is furtherdesirable to selectively produce, via a biochemical pathway, cis-isomersof the alcohols such as iso-α-sinensol, iso-β-sinensol, (Z)-α-santalol,(Z)-β-santalol, (Z)-α-trans-bergamotol and (Z)-epi-β-santalol.

Cytochrome P450s represent a family of enzymes of oxidases. P450scommonly catalyze a monooxygenase reaction. Cytochrome P450 enzymes areclassified into families and subfamilies based on the amino acidsequences homology. Members of a same subfamily share over 55% aminoacid sequence identity and have usually similar enzymatic activities(substrate and/or product selectivity). CYP71AV1 (NCBI accession NoABB82944.1, SEQ ID No. 51 and 52) and CYP71AV8 (NCBI accession NoADM86719.1, SEQ ID No. 1 and 2) are two members of the CYP71AVsub-family and shares 78% sequence identity. CYP71AV1 has previouslybeen shown to oxidize amorphadiene (Teoh et al, FEBS letters 580 (2006)1411-1416). CYP71AV8 has previously been shown to oxidize (+)-valencene,germacrene A and amorphadiene (Cankar et al, FEBS Lett. 585(1), 178-182(2011)).

Processes using engineered cells have been reported that use terpenesynthases to catalyze the production of a diterpene or sesquiterpene.The diterpenes or sesquiterpenes were further processed using acytochrome P450 polypeptide to catalyze the hydroxylation, oxidation,demethylation or methylation of the diterpene or sesquiterpene producedby the cell.

SUMMARY

Provided herein is a method of producing an sesquiterpene alcoholcomprising:

i) contacting a terpene of Formula I:

with a polypeptide having an amino acid sequence having at least, or atleast about, 45% of sequence identify to a polypeptide selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 79, and SEQ ID NO: 81; and

ii) optionally isolating the alcohol wherein R is a saturated,mono-unsaturated or poly-unsaturated aliphatic group composed of 9carbons and wherein R can be a branched chain or composed of one or morenon-aromatic rings.

Further provided herein is a method of producing a sesquiterpenecomprising α-sinensol, β-sinensol, α-santalol, β-santalol,α-trans-bergamotol, epi-β-santalol, lancelol and/or or mixtures thereofcomprising:

i) contacting α-farnesene, β-farnesene, α-santalene, β-santalene,α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene, with apolypeptide having an amino acid sequence having at least, or at leastabout, 45% of sequence identify to a polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71,SEQ ID NO: 73, SEQ ID NO: 79, and SEQ ID NO: 81 to produce the alcohol;and

ii) optionally isolating the alcohol.

Also provided herein is a method of producing α-sinensol, β-sinensol,α-santalol, β-santalol, α-trans-bergamotol, epi-β-santalol, lanceloland/or mixtures thereof comprising contacting α-farnesene, β-farnesene,α-santalene, β-santalene, α-trans-bergamotene and/or epi-β-santalene,with a polypeptide having a P450 monooxygenase activity wherein thesesquiterpene alcohol produced comprises at least, or at least about,36% of a cis isomer.

Further provided herein is an isolated polypeptide having monooxygenaseactivity comprising an amino acid sequence that is at least, or at leastabout 45%, 50%, 55%, 50%, 65%, 70%, 80%, 90%, 95%, 98% or more identicalto an amino acid sequence selected from the group consisting of SEQ IDNO: 71, and SEQ ID NO:73.

Further provided herein is an isolated polypeptide having monooxygenaseactivity comprising an amino acid sequence that is at least, or at leastabout 45%, 50%, 55%, 50%, 65%, 70%, 80%, 90%, 95%, 98% or more identicalto an amino acid sequence selected from the group consisting of SEQ IDNO: 79, and SEQ ID NO: 81.

Also provided herein is an isolated polypeptide having monooxygenaseactivity comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQID NO: 34, SEQ ID NO:36, SEQ ID NO:71, SEQ ID NO:73 SEQ ID NO: 79, andSEQ ID NO: 81.

Further provided herein is method of producing a sesquiterpene alcoholselected from the group consisting of α-sinensol, β-sinensol,α-santalol, β-santalol, α-trans-bergamotol, epi-β-santalol, and lancelolor mixtures thereof:

i) cultivating a cell under conditions suitable to produce a p450polypeptide having monooxygenase activity wherein the cell: a) producesa acylic pyrophosphate terpene precursor; b) expresses a P450 reductase,c) expresses a polypeptide that has α-farnesene, β-farnesene,α-santalene, β-santalene, α-trans-bergamotene and/or epi-β-santalene,synthase activity and produces α-farnesene, β-farnesene, α-santalene,β-santalene, α-trans-bergamotene and/or epi-β-santalene and d) expressesa polypeptide with an amino acid sequence having at least, or at leastabout, 45% of sequence identify to a polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71,SEQ ID NO: 73 SEQ ID NO: 79, and SEQ ID NO: 81; and

ii) optionally isolating the alcohol from the cell.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence alignment of the N-terminal region of thedifferent CYP71AV8 variants: CYP71AV8_wt (SEQ ID NO: 2), cyp71AV8-65188(SEQ ID NO: 4), CYP71AV8-P2 (SEQ ID NO: 6), CYP71AV8-P20 (SEQ ID NO: 8).

FIG. 2A-D. Alignment of DNA sequences of the different CYP71AV8variants: CYP71AV8_wt (SEQ ID NO: 1), cyp71AV8-65188 (SEQ ID NO: 3),CYP71AV8-P2 (SEQ ID NO: 5), CYP71AV8-P20 (SEQ ID NO: 6). The encodedamino acid sequences are shown below each sequence using the one-lettercode.

FIG. 3. GCMS analysis of the conversion of sesquiterpenes by E. colicells expressing the CYP71AV8 and the CPRm proteins. A, Bioconversion of(+)-alpha-santalene. B, Bioconversion of a(+)-alpha-santalene/(−)-beta-santalene mixture.

FIG. 4. Organisation of the synthetic bi-cistronic operon containing aP450 and a CPR cDNA.

FIG. 5. Comparison of the bioconversion of (+)-α-santalene and theα/β-santalene mixture by E. coli cells transformed with differentbi-cistronic operons composed of a P450 and a CPR cDNA. 1,CYP71AV8-65188 and aaCPR. 2, CYP71AV8-P2 and aaCPR. 3, CYP71AV8-P2O andaaCPR. 4, CYP71AV8-65188 and CPRm. 5, CYP71AV8-P2 and CPRm. 6,CYP71AV8-P2O and CPRm.

FIG. 6. GCMS analysis of the sesquiterpene molecules produced by E. colicells expressing CYP71AV8, CPRm, an alpha-santalene synthase (A) or aalpha-santalene/beta-santalene synthase (B), and mevalonate pathwayenzymes. 1, (+)-α-santalene; 2 (−)-α-trans-bergamotene; 3,(+)-epi-β-santalene; 4, (−)-β-santalene.

FIG. 7. Oxidation of (+)-α-santalene by CYP71AV8 wild type (A) andmutant L-358 (B). GC-MS profiles of the sesquiterpene products generatedby E. coli KRX cells expressing CPRm, ClASS, the mevalonate pathwayenzymes and CYP71AV8 (A) or CYP71AV8-L358F (B). The cultivations wereperformed in TB medium containing 3% glycerol as carbon source. Thedifferent products were identified as α-santalene (1),(E)-α-santalal(2), (Z)-α-santalol (4), and (E)-α-santalol (3).

FIG. 8. GC-MS profiles of the sesquiterpene products generated by E.coli KRX cells expressing CPRm, SaSAS, the mevalonate pathway enzymesand CYP71AV8-L358F. The cultivations were performed in TB mediumcontaining 3% glycerol as carbon source. The different productsidentified by their mass spectra are indicated.

FIG. 9. GCMS analysis of the conversion of (+)-α-santalene by E. colicells expressing the CYP71AV1 and the CPRm proteins.

FIG. 10: GC analysis of the in vivo conversion of (+)-α-santalene to(Z)-α-santalol by a P450-BM3 double-mutant (variant #17). Solventextracts of cultures of recombinant E. coli cells co-expressing aClausena lansium α-santalene synthase and either the wild-type P450-BM3(A) or the P450-BM3 variant #17 (B) were analyzed as described inexample 11. 1, (+)-α-santalene; 2, (−)-α-trans-bergamotene; 3,(Z)-α-santalol; The chromatograms are shown in selected ion mode (M/Z93).

FIG. 11: GC analysis of the in vivo conversion of (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene and (+)-epi-β-santalene by aP450-BM3 double-mutant. Solvent extracts of cultures of recombinant E.coli cells co-expressing an alpha-santalene/beta-santalene synthase fromSantalum album and either the wild-type P450-BM3 (A) or the P450-BM3variant #17 (B) were analyzed as described in example 11. 1,(+)-α-santalene; 2, (−)-α-trans-bergamotene; 3, (+)-epi-β-santalene; 4,(−)-β-santalene; 5, (Z)-α-santalol; 6, (Z)-α-trans-bergamotol; 7,(Z)-epi-β-santalol; 8, (Z)-β-santalol. The chromatograms are shown inselected ion (M/Z 93).

FIG. 12: GCMS analysis of the conversion of (+)-α-santalene by therecombinant SaCP816 enzyme. A. Control without the recombinant P450enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP816 protein. C. Sandalwood oil for comparison of theretention times. All assays were performed in-vitro as described inexample 4. 1, (+)-α-santalene; 5, (Z)-α-santalol; 6,(Z)-α-trans-bergamotol; 7, (Z)-epi-β-santalol; 8, (Z)-β-santalol. Theidentity of the sequiterpene molecules were confirmed by matching of themass spectra with authentic standards.

FIG. 13: GCMS analysis of the conversion of (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene and (+)-epi-β-santalene by therecombinant SaCP816 enzyme. A. Control without the recombinant P450enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP816 protein. C. Sandalwood oil for comparison of theretention times. All assays were performed in-vitro as described inexample 4. 1, (+)-α-santalene; 2, (−)-α-trans-bergamotene; 3,(+)-epi-β-santalene; 4, (−)-β-santalene; 5, (Z)-α-santalol; 6,(Z)-α-trans-bergamotol; 7, (Z)-epi-β-santalol; 8, (Z)-β-santalol. Theidentity of the sequiterpene molecules were confirmed by matching of themass spectra with authentic standards.

FIG. 14: GCMS analysis of the molecules produced by E. coli engineeredto produced sesquiterpenes and expressing SaCP816, CPRm, analpha-santalene synthase (ClASS) (A) or a alpha-santalene/beta-santalenesynthase (SaSAS) (B). 1, (+)-α-santalene; 2, (−)-α-trans-bergamotene; 3,(+)-epi-β-santalene; 4, (−)-β-santalene; 5, (Z)-α-santalol; 6,(Z)-α-trans-bergamotol; 7, (Z)-epi-β-santalol; 8, (Z)-β-santalol(co-eluted with farnesol produced from an excess pool of farnesyldiphosphate). The identity of the sequiterpene molecules were confirmedby matching of the mass spectra with authentic standards.

FIG. 15: GCMS analysis of the conversion of (+)-α-santalene (21) by therecombinant SaCP10374 P450 enzyme. A. Control without the recombinantP450 enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP10374 protein. The numbers indicated on thechromatograms refer to the structures presented in FIG. 27.

FIG. 16: GCMS analysis of the conversion of a mixture composed of(+)-α-santalene (21), (−)-α-trans-bergamotene (17); (+)-epi-β-santaleneand (−)-β-santalene (25) (prepared using the SaTp8201 recombinantprotein, example 4) by the recombinant SaCP10374 P450s enzymes. A.Control without the recombinant P450 enzyme. B. Assay with E. coli crudeprotein extract containing the recombinant SaCP10374 protein. Thenumbers indicated on the chromatograms refer to the structures presentedin FIG. 27.

FIG. 17: GCMS analysis of the conversion of β-farnesene (1) by therecombinant S. album P450s enzymes. A. Control without the recombinantP450 enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP10374 protein. C. Assay with E. coli crude proteinextract containing the recombinant SaCP816 protein. The numbersindicated on the chromatograms refer to the structures presented in FIG.27.

FIG. 18: GCMS analysis of the conversion of α-farnesene (5) by therecombinant S. album P450s enzymes. A. Control without the recombinantP450 enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP10374 protein. C. Assay with E. coli crude proteinextract containing the recombinant SaCP816 protein. The numbersindicated on the chromatograms refer to the structures presented in FIG.27.

FIG. 19: GCMS analysis of the conversion of (−)-sesquisabinene B (9) bythe recombinant S. album P450s enzymes. A. Control without therecombinant P450 enzyme. B. Assay with E. coli crude protein extractcontaining the recombinant SaCP10374 protein. C. Assay with E. colicrude protein extract containing the recombinant SaCP816 protein. Thenumbers indicated on the chromatograms refer to the structures presentedin FIG. 27.

FIG. 20: GCMS analysis of the conversion of (−)-β-bisabolene (13) by therecombinant S. album P450s enzymes. A. Control without the recombinantP450 enzyme. B. Assay with E. coli crude protein extract containing therecombinant SaCP10374 protein. C. Assay with E. coli crude proteinextract containing the recombinant SaCP816 protein. The numbersindicated on the chromatograms refer to the structures presented in FIG.27.

FIG. 21: GCMS analysis of the conversion of (−)-α-bergamotene (17) bythe recombinant S. album P450s enzymes. A. Control without therecombinant P450 enzyme. B. Assay with E. coli crude protein extractcontaining the recombinant SaCP10374 protein. C. Assay with E. colicrude protein extract containing the recombinant SaCP816 protein. Thenumbers indicated on the chromatograms refer to the structures presentedin FIG. 27.

FIG. 22: GCMS analysis of the products generated in-vivo as described inexample 23 by E. coli KRX cells transformed with the plasmidspACYC-29258-4506 and the plasmid pD444-SR-AaBFS (A),SaCP10374-CPRm-AaBFS-pCWori (B), or SaCP816-CPRm-AaBFS-pCWori (C). Thechromatograms show the formation of (E)-β-farnesene (1) as well asoxidized derivatives (2-3) (see FIG. 27 for corresponding structures).

FIG. 23: GCMS analysis of the products generated in-vivo as described inexample 23 by E. coli KRX cells transformed with the plasmidspACYC-29258-4506 and the plasmid pD444-SR-PaBAFS (A),SaCP10374-CPRm-PaAFS-pCWori (B), or SaCP816-CPRm-PaAFS-pCWori (C). Thechromatograms show the formation of (E,E)-α-farnesene (5) as well asoxidized derivatives (6-8) (see FIG. 27 for corresponding structures).The peak of farnesol resulting from the hydrolysis of excess FPP isinducated on each chromatogram.

FIG. 24: GCMS analysis of the products generated in-vivo as described inexample 23 by E. coli KRX cells transformed with the plasmidspACYC-29258-4506 and the plasmid pETDuet-SaTps647 (A),SaCP10374-CPRm-SaTps647-pCWori (B), or SaCP816-CPRm-SaTPS647-pCWori(C).The chromatograms show the formation of (−)-sesquisabinene B (9) as wellas oxidized derivatives (10-12) (see FIG. 27 for correspondingstructures).

FIG. 25: GCMS analysis of the products generated in-vivo as described inexample 23 by E. coli KRX cells transformed with the plasmidspACYC-29258-4506 and the plasmid pETDuet-ClTps2 (A) orSaCP10374-CPRm-ClTps2-pCWori (B). The chromatograms show the formationof (+)-α-santalene (21) as well as oxidized derivatives (23-24) (seeFIG. 27 for corresponding structures).

FIG. 26: GCMS analysis of the products generated in-vivo as described inexample 23 by E. coli KRX cells transformed with the plasmidspACYC-29258-4506 and the plasmid pETDuet-SaTps8201 (A) orSaCP10374-CPRm-SaTps8201-pCWori (B). The chromatograms show theformation of (+)-α-santalene (21), (−)-β-santalene (25) and(−)-trans-α-Bergamotene (17) as well as oxidized derivatives (19, 20,23, 24, 27 and 28) (see FIG. 27 for corresponding structures).

FIG. 27A-B: Structure of the enzymes substrates and products discussedin the text.

DETAILED DESCRIPTION

In some embodiments, provided herein is a method of producing asesquiterpene comprising α-sinensol, β-sinensol, α-santalol, β-santalol,α-trans-bergamotol, epi-β-santalol, and lancelol and/or mixtures thereofcomprising contacting α-farnesene, farnesene, α-santalene, β-santalene,α-trans-bergamotene and/or epi-β-santalene, with a polypeptidecomprising an amino acid sequence having at least, or at least about,45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify toSEQ ID NO: 2. In a particular embodiment, the method comprises a cellthat expresses the polypeptide.

In some embodiments, provided herein is a method of producing aα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 4. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 6. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 8. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 28. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 30. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 32. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 34. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 36. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 38. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 40. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 42. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 44. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 50. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 52. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 54. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 58. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO:60. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 62. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 64. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 66. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 68. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 71. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 73. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 79. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

In some embodiments, provided herein is a method of producingα-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereof comprising contactingα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamoteneand/or epi-β-santalene, with a polypeptide comprising an amino acidsequence having at least, or at least about, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 95%, or 98% sequence identify to SEQ ID NO: 81. In aparticular embodiment, the method comprises a cell that expresses thepolypeptide.

The nucleotide sequences provided herein for producing a polypeptide foruse in producing an alcohol have a nucleic acid sequence at least, or atleast about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, SEQ ID NO: 67, SEQ ID NO: 70 SEQ ID NO: 72, SEQ ID NO: 78 and SEQ IDNO: 80. The nucleotide sequences provided herein are heterologous inthat they are not typically or normally produced by a cell in which itis expressed herein and is generally not endogenous to the cell intowhich it is introduced—it being typically obtained from another cell orcould be made synthetically.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 36%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 71, and SEQ ID NO: 73.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 46%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 50%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:68, SEQ ID NO: 71, and SEQ ID NO: 73.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 72%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98%sequence identify to a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 96%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73.

In another embodiment, provided herein is a method of producing asesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol,β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol, and/ormixtures thereof comprising contacting trans-α-farnesene transβ-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450monoxygenase activity wherein the alcohol produced comprises at least,or at least about, 100%, of a cis isomer and wherein the polpeptide ecomprises an amino acid sequence having at least or at least about 45%,50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identify to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 71, and 73.

Provided herein is also an isolated nucleic acid molecule selected fromthe group consisting of: i) a nucleic acid having an nucleic acidsequence selected from the group consisting SEQ ID. NO: 70 and 72; andii) a nucleic acid molecule that encodes a polypeptide having p450monooxygenase activity wherein the polypeptide comprises an amino acidsequence that is at least, or at least about 45%, 50%, 55%, 50%, 65%,70%, 80%, 90%, 95%, or 98% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 71, and SEQ ID NO: 73.More particularly the polypeptide encoded has the sequence selected fromthe group consisting of SEQ ID NOs: 71, and SEQ ID NO: 73.

Provided herein is also an isolated nucleic acid molecule selected fromthe group consisting of: i) a nucleic acid having an nucleic acidsequence selected from the group consisting SEQ ID. NO: 78 and 80; andii) a nucleic acid molecule that encodes a polypeptide having p450monooxygenase activity wherein the polypeptide comprises an amino acidsequence that is at least, or at least about 45%, 50%, 55%, 50%, 65%,70%, 80%, 90%, 95%, or 98% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 79, and SEQ ID NO: 82.More particularly the polypeptide encoded has the sequence selected fromthe group consisting of SEQ ID NOs: 79, and SEQ ID NO: 82.

Also provided herein is an isolated nucleic acid molecule selected fromthe group consisting of: i) a nucleic acid having an nucleic acidsequence selected from the group consisting SEQ ID. NO: 27, 29, 31, 33,and 35; and ii) a nucleic acid molecule that encodes a polypeptidehaving p450 monooxygenase activity wherein the polypeptide has thesequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36.

In another embodiment provided herein is a method for producing apolypeptide having P450 monoxygenase activity comprising the steps oftransforming a host cell or non-human organism with a nucleic acidencoding a polypeptide having at least, or at least about, 45%, 50%,55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identity to apolypeptide selected from the group consisting of SEQ ID NO: 71, and SEQID NO: 73 and culturing the host cell or organism under conditions thatallow for the production of the polypeptide.

In a further embodiment provided here is a method for producing apolypeptide having P450 monoxygenase activity comprising the steps oftransforming a host cell or non-human organism with a nucleic acidencoding a polypeptide having the sequence selected from the groupconsisting of SEQ ID NO: 71, and SEQ ID NO: 73 and culturing the hostcell or organism under conditions that allow for the production of thepolypeptide.

In another embodiment provided herein is a method for producing apolypeptide having P450 monoxygenase activity comprising the steps oftransforming a host cell or non-human organism with a nucleic acidencoding a polypeptide having at least, or at least about, 45%, 50%,55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identity to apolypeptide selected from the group consisting of SEQ ID NO: 79, and SEQID NO: 81 and culturing the host cell or organism under conditions thatallow for the production of the polypeptide.

In a further embodiment provided here is a method for producing apolypeptide having P450 monoxygenase activity comprising the steps oftransforming a host cell or non-human organism with a nucleic acidencoding a polypeptide having the sequence selected from the groupconsisting of SEQ ID NO: 79, and SEQ ID NO: 81 and culturing the hostcell or organism under conditions that allow for the production of thepolypeptide.

The alcohols can be converted to aldehydes or acids such as but notlimited to sinensals, santalals, bergamotenals, and lanceals. Thealcohols, aldehydes or acids can be further converted to derivativessuch as, but not limited to esters, amides, glycosides, ethers oracetals.

Nucleic acid and polypeptides described herein may be isolated forexample from Cichorium intybus L., Bacillus megaterium, Santalum albumand Artemisia annua. CYP71AV8, P450-BM3 (CYP102A1), and CYP71AV1including variants are described herein.

CYP71AV8 from the plant Cichorium intybus L. was previouslycharacterized as a P450 mono-oxygenase able to oxidizeregion-selectively (+)-valencene producing trans-nootkatol,cis-nootkatol and (+)-nootkatone. CYP71AV8 was also found to catalysethe oxidation of germacrene A and amorpha-4,11-diene in the C-12position (Cankar et al, FEBS Lett. 585(1), 178-182 (2011)). The aminoacid sequence of the wild type enzyme (NCBI accession No ADM86719.1, SEQID No 1 and 2) was used to design a cDNA sequence optimized forexpression in E. coli.

In eukaryotes, the P450 monooxygenases are membrane-bound proteins andthe N-terminal sequence of these proteins constitute a membrane anchoressential for the membrane localization of these enzymes. This part ofthe protein, usually delimited by a proline-rich domaine, is notessential for the control of the specificity of the enzymatic activity.This region can thus be modified by deletion, insertion or mutationwithout effect on the catalytic activity. However, specific modificationof the N-terminal region of eukaryotic P450s, including plant P450s,have been shown to have a positive effect on the levels of functionalrecombinant proteins when expressed in microorganisms (Halkier et al(1995) Arch. Biochem. Biophys. 322, 369-377; Haudenschield et al (2000)Arch. Biochem. Biophys. 379, 127-136).

In P450 monooxygenases the recognition and binding of the substrate iscontrolled by several amino acid residues distributed in differentregions along the protein amino acid sequences. These regions, definedas substrate recognition sites (SRS), can be localized in the amino acidsequence of any P450 by simple sequence alignment based for example onthe work made by Gotoh (Gotoh O (1992) J. Biol. Chem. 267(1), 83-90).Thus residues in the CYP71AV8 protein that interact with the substrateand can influence the regioselectivity of the hydroxylation reaction arethe amino acids Asn98 to Gly121, Thr198 to Leu205, Lys232 to Ile 240,Asn282 to Ala300, His355 to Arg367 and Thr469 to Val 476. Themodification of one or more residues in these regions can potentiallyalter the substrate specificity, the stereochemistry of the reaction orits regioselectivity. One example of alteration of the regioselectivityof the reaction catalyzed by a P450 can be found in Schalk et al (2002)Proc. Natl. Acad. Sci. USA 97(22), 11948-11953. In this publication asingle residue change in plant P450 enzymes led to a complete conversionto the regiospecificity of the enzymatique reaction.

A “sesquiterpene synthase” or a “polypeptide having a sesquiterpenesynthase activity” is intended for the purpose of the presentapplication as a polypeptide capable of catalyzing the synthesis of asesquiterpene molecule or of a mixture of sesquiterpene molecules from aacyclic pyrophosphate terpene precursor selected from the groupconsisting of geranyl-pyrophosphate (GPP), farnesy-diphosphate (FPP) andgeranylgeranyl-pyrophosphate (GGPP).

Alpha santalene, beta-santalene, alpha-trans-bergamotene, and/orepi-beta santalene may be prepared using the synthases described forexample in U.S. Patent Publication No.: 2011-0008836, published Jan. 13,20111 and in U.S. Patent Publication No.: 2011-0281257, published Nov.27, 2011, both of which are incorporated herein in their entirety.

According to the present invention, polypeptides are also meant toinclude truncated polypeptides provided that they keep their P450monooxygenase activity as defined in any of the above embodiments.

The percentage of identity between two peptidic or nucleotidic sequencesis a function of the number of amino acids or nucleotide residues thatare identical in the two sequences when an alignment of these twosequences has been generated. Identical residues are defined as residuesthat are the same in the two sequences in a given position of thealignment. The percentage of sequence identity, as used herein, iscalculated from the optimal alignment by taking the number of residuesidentical between two sequences dividing it by the total number ofresidues in the shortest sequence and multiplying by 100. The optimalalignment is the alignment in which the percentage of identity is thehighest possible. Gaps may be introduced into one or both sequences inone or more positions of the alignment to obtain the optimal alignment.These gaps are then taken into account as non-identical residues for thecalculation of the percentage of sequence identity.

Alignment for the purpose of determining the percentage of amino acid ornucleic acid sequence identity can be achieved in various ways usingcomputer programs and for instance publicly available computer programsavailable on the world wide web. Particularly, the BLAST program(Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set tothe default parameters, available from the National Center forBiotechnology Information (NCBI) at their webpagencbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain anoptimal alignment of peptidic or nucleotidic sequences and to calculatethe percentage of sequence identity.

A particular organism or cell is meant to be “capable of producing FPP”when it produces FPP naturally or when it does not produce FPP naturallybut is transformed to produce FPP, either prior to the transformationwith a nucleic acid as described herein or together with said nucleicacid. Organisms or cells transformed to produce a higher amount of FPPthan the naturally occurring organism or cell are also encompassed bythe “organisms or cells capable of producing FPP”. Methods to transformorganisms, for example microorganisms, so that they produce FPP arealready known in the art. Such methods can for example be found in theliterature, for example in the following publications: Martin, V. J.,Pitera, D. J., Withers, S. T., Newman, J. D., and Keasling, J. D. NatBiotechnol., 2003, 21(7), 796-802 (transformation of E. coli); Wu, S.,Schalk, M., Clark, A., Miles, R. B., Coates, R., and Chappell, J., NatBiotechnol., 2006, 24(11), 1441-1447 (transformation of plants);Takahashi, S., Yeo, Y., Greenhagen, B. T., McMullin, T., Song, L.,Maurina-Brunker, J., Rosson, R., Noel, J., Chappell, J, Biotechnologyand Bioengineering, 2007, 97(1), 170-181 (transformation of yeast).

Non-human host organisms suitable to carry out the method describedherein in vivo may be any non-human multicellular or unicellularorganisms. In a particular embodiment, the non-human host organism usedto carry out the invention in vivo is a plant, a prokaryote or a fungus.Any plant, prokaryote or fungus can be used. Particularly useful plantsare those that naturally produce high amounts of terpenes. In a moreparticular embodiment, the plant is selected from the family ofSolanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae orLamiaceae. For example, the plant is selected from the genera Nicotiana,Solanum, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa),Gossypium (cotton), Artemisia, Salvia and Mentha. Particularly, theplant belongs to the species of Nicotiana tabacum.

In a more particular embodiment the non-human host organism used tocarry out the method of the invention in vivo is a microorganism. Anymicroorganism can be used but according to an even more particularembodiment said microorganism is a bacteria or yeast. Most particularly,said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

Some of these organisms do not produce FPP naturally. To be suitable tocarry out the method of the invention, these organisms have to betransformed to produce said precursor. They can be so transformed eitherbefore the modification with the nucleic acid described according to anyof the above embodiments or simultaneously, as explained above.

Isolated higher eukaryotic cells can also be used, instead of completeorganisms, as hosts to carry out the method of the invention in vivo.Suitable eukaryotic cells may be any non-human cell, but areparticularly plant or fungal cells.

As used herein, the polypeptide is intended as a polypeptide or peptidefragment that encompasses the amino acid sequences identified herein, aswell as truncated or variant polypeptides, provided that they keep theirP450 monooxygenaseactivity as defined above and that they share at leastthe defined percentage of identity with the corresponding polypeptide.

Examples of variant polypeptides are naturally occurring proteins thatresult from alternate mRNA splicing events or from proteolytic cleavageof the polypeptides described herein. Variations attributable toproteolysis include, for example, differences in the N- or C-terminiupon expression in different types of host cells, due to proteolyticremoval of one or more terminal amino acids from the polypeptides of theinvention. Polypeptides encoded by a nucleic acid obtained by natural orartificial mutation of a nucleic acid of the invention, as describedthereafter, are also encompassed by the invention.

Polypeptide variants resulting from a fusion of additional peptidesequences at the amino and carboxyl terminal ends can also be used inthe methods of the invention. In particular such a fusion can enhanceexpression of the polypeptides, be useful in the purification of theprotein or improve the enzymatic activity of the polypeptide in adesired environment or expression system. Such additional peptidesequences may be signal peptides, for example. Accordingly, the presentinvention encompasses methods using variant polypeptides, such as thoseobtained by fusion with other oligo- or polypeptides and/or those whichare linked to signal peptides. Polypeptides resulting from a fusion withanother functional protein, such as another protein from the terpenebiosynthesis pathway, can also be advantageously be used in the methodsof the invention.

As used herein, the polypeptide is intended as a polypeptide or peptidefragment that encompasses the amino acid sequence identified herein, aswell as truncated or variant polypeptides, provided that they keep theiractivity as defined above. Examples of variant polypeptides arenaturally occurring proteins that result from alternate mRNA splicingevents or from proteolytic cleavage of the polypeptides describedherein. Variations attributable to proteolysis include, for example,differences in the N- or C-termini upon expression in different types ofhost cells, due to proteolytic removal of one or more terminal aminoacids from the polypeptides of the invention. Polypeptides encoded by anucleic acid obtained by natural or artificial mutation of a nucleicacid of the invention, as described thereafter, are also encompassed bythe invention.

Polypeptide variants resulting from a fusion of additional peptidesequences at the amino and carboxyl terminal ends are also encompassedby the polypeptides of the invention. In particular such a fusion canenhance expression of the polypeptides, be useful in the purification ofthe protein or improve the enzymatic activity of the polypeptide in adesired environment or expression system. Such additional peptidesequences may be signal peptides, for example. Accordingly, the presentinvention encompasses variants of the polypeptides of the invention,such as those obtained by fusion with other oligo- or polypeptidesand/or those which are linked to signal peptides. Polypeptides resultingfrom a fusion with another functional protein, such as another proteinfrom the terpene biosynthesis pathway, are also encompassed by thepolypeptides of the invention.

The nucleic acid of the invention can be defined as includingdeoxyribonucleotide or ribonucleotide polymers in either single- ordouble-stranded form (DNA and/or RNA). The terms “nucleotide sequence”should also be understood as comprising a polynucleotide molecule or anoligonucleotide molecule in the form of a separate fragment or as acomponent of a larger nucleic acid. Nucleic acids of the invention alsoencompass certain isolated nucleotide sequences including those that aresubstantially free from contaminating endogenous material. The nucleicacid of the invention may be truncated, provided that it encodes apolypeptide encompassed by the present invention, as described above.

Another important tool for transforming host organisms or cells suitableto carry out the method of the invention in vivo is an expression vectorcomprising a nucleic acid according to any embodiment of the invention.Such a vector is therefore also an object of the present invention.

An “expression vector” as used herein includes any linear or circularrecombinant vector including but not limited to viral vectors,bacteriophages and plasmids. The skilled person is capable of selectinga suitable vector according to the expression system. In one embodiment,the expression vector includes the nucleic acid of the inventionoperably linked to at least one regulatory sequence, which controlstranscription, translation, initiation and termination, such as atranscriptional promoter, operator or enhancer, or an mRNA ribosomalbinding site and, optionally, including at least one selection marker.Nucleotide sequences are “operably linked” when the regulatory sequencefunctionally relates to the nucleic acid of the invention.

The expression vectors of the present invention may be used in themethods for preparing a genetically transformed host organism and/orcell, in host organisms and/or cells harboring the nucleic acids of theinvention and in the methods for making polypeptides having a P450monooxygenase activity, as disclosed further below.

Recombinant non-human host organisms and cells transformed to harbor atleast one nucleic acid of the invention so that it heterologouslyexpresses or over-expresses at least one polypeptide of the inventionare also very useful tools to carry out the method of the invention.Such non-human host organisms and cells are therefore another object ofthe present invention.

A nucleic acid according to any of the above-described embodiments canbe used to transform the non-human host organisms and cells and theexpressed polypeptide can be any of the above-described polypeptides.

Non-human host organisms of the invention may be any non-humanmulticellular or unicellular organisms. In a particular embodiment, thenon-human host organism is a plant, a prokaryote or a fungus. Any plant,prokaryote or fungus is suitable to be transformed according to thepresent invention. Particularly useful plants are those that naturallyproduce high amounts of terpenes. In a more particular embodiment, theplant is selected from the family of Solanaceae, Poaceae, Brassicaceae,Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant isselected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis,Brassica (rape), Medicago (alfalfa), Gossypium (cotton), Artemisia,Salvia and Mentha. Particularly, the plant belongs to the species ofNicotiana tabacum.

In a more particular embodiment the non-human host organism is amicroorganism. Any microorganism is suitable for the present invention,but according to an even more particular embodiment said microorganismis a bacteria or yeast. Most particularly, said bacteria is E. coli andsaid yeast is Saccharomyces cerevisiae.

Isolated higher eukaryotic cells can also be transformed, instead ofcomplete organisms. As higher eukaryotic cells, we mean here anynon-human eukaryotic cell except yeast cells. Particular highereukaryotic cells are plant cells or fungal cells.

The term “transformed” refers to the fact that the host was subjected togenetic engineering to comprise one, two or more copies of each of thenucleic acids required in any of the above-described embodiment.Particularly the term “transformed” relates to hosts heterologouslyexpressing the polypeptides encoded by the nucleic acid with which theyare transformed, as well as over-expressing said polypeptides.Accordingly, in an embodiment, the present invention provides atransformed organism, in which the polypeptides are expressed in higherquantity than in the same organism not so transformed.

There are several methods known in the art for the creation oftransgenic host organisms or cells such as plants, fungi, prokaryotes,or cultures of higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, plant andmammalian cellular hosts are described, for example, in Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1985, Elsevier, New York andSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,1989, Cold Spring Harbor Laboratory Press. Cloning and expressionvectors for higher plants and/or plant cells in particular are availableto the skilled person. See for example Schardl et al. Gene 61: 1-11,1987.

Methods for transforming host organisms or cells to harbor transgenicnucleic acids are familiar to the skilled person. For the creation oftransgenic plants, for example, current methods include: electroporationof plant protoplasts, liposome-mediated transformation,agrobacterium-mediated transformation, polyethylene-glycol-mediatedtransformation, particle bombardment, microinjection of plant cells, andtransformation using viruses.

In one embodiment, transformed DNA is integrated into a chromosome of anon-human host organism and/or cell such that a stable recombinantsystem results. Any chromosomal integration method known in the art maybe used in the practice of the invention, including but not limited torecombinase-mediated cassette exchange (RMCE), viral site-specificchromosomal insertion, adenovirus and pronuclear injection.

A “polypeptide variant” as referred to herein means a polypeptide havingthe above described activity and being substantially homologous to thepolypeptide according to any of the above embodiments, but having anamino acid sequence different from that encoded by any of the nucleicacid sequences of the invention because of one or more deletions,insertions or substitutions.

Variants can comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.See Zubay, Biochemistry, 1983, Addison-Wesley Pub. Co. The effects ofsuch substitutions can be calculated using substitution score matricessuch a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, J. Mol.Biol., 1991, 219, 555-565. Other such conservative substitutions, forexample substitutions of entire regions having similar hydrophobicitycharacteristics, are well known.

Naturally occurring peptide variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of thepolypeptides described herein. Variations attributable to proteolysisinclude, for example, differences in the N- or C-termini upon expressionin different types of host cells, due to proteolytic removal of one ormore terminal amino acid from the polypeptides encoded by the sequencesof the invention.

Variants of the polypeptides of the invention may be used to attain forexample desired enhanced or reduced enzymatic activity, modifiedregiochemistry or stereochemistry, or altered substrate utilization orproduct distribution, increased affinity for the substrate, improvedspecificity for the production of one or more desired compounds,increased velocity of the enzyme reaction, higher activity or stabilityin a specific environment (pH, temperature, solvent, etc), or improvedexpression level in a desired expression system. A variant or sitedirected mutant may be made by any method known in the art. Variants andderivatives of native polypeptides can be obtained by isolatingnaturally-occurring variants, or the nucleotide sequence of variants, ofother or same plant lines or species, for examples plants from theSantalum species, or by artificially programming mutations of nucleotidesequences coding for the polypeptides of the invention. Alterations ofthe native amino acid sequence can be accomplished by any of a number ofconventional methods.

Polypeptide variants resulting from a fusion of additional peptidesequences at the amino and carboxyl terminal ends of the polypeptides ofthe invention can be used to enhance expression of the polypeptides, beuseful in the purification of the protein or improve the enzymaticactivity of the polypeptide in a desired environment or expressionsystem. Such additional peptide sequences may be signal peptides, forexample. Accordingly, the present invention encompasses variants of thepolypeptides of the invention, such as those obtained by fusion withother oligo- or polypeptides and/or those which are linked to signalpeptides. Fusion polypeptide encompassed by the invention also comprisefusion polypeptides resulting from a fusion of other functionalproteins, such as other proteins from the terpene biosynthesis pathway.

The alcohols produced herein may be isolated by extraction for exampleusing known methods to extract the alcohols generated in nature (e.g.,extraction from Sandalwood). The alcohols produced herein have use asfragrant compounds that may be used in perfumery.

ABBREVIATIONS USED

-   aaCPR Arthemisia annua Cytochrome P450 reductase-   bp base pair-   kb kilo base-   DNA deoxyribonucleic acid-   cDNA complementary DNA-   ClASS Clausena lansium (+)-α-santalene synthase-   CPRm Mentha piperita Cytochrome P450 reductase-   DTT dithiothreitol-   EDTA ethylene-diamine-tetraacetic acid-   FPP farnesyl pyrophosphate-   GC gaseous chromatograph-   IPTG isopropyl-D-thiogalacto-pyranoside-   LB lysogeny broth-   MS mass spectrometer-   MTBE methyl tert-buthyl ether-   PCR polymerase chain reaction-   RMCE recombinase-mediated cassette exchange-   RNA ribonucleic acid-   mRNA messenger ribonucleic acid-   SaSAS Santalum album (+)-α-santalene/(⊕)-β-santalene synthase

The following examples are illustrative only and are not meant to limitthe scope of invention as set forth in the Summary, Description or inthe Claims.

EXAMPLES Example 1 Optimization of the CYP71AV8 cDNA sequence forexpression in bacteria

The membrane anchor region of CYP71AV8 was redesigned to introduce themodifications detailed bellow.

In the optimized CYP71AV8 sequences the 5′-end was modified to replacethe first amino acids of the membrane anchor region with a peptidesequence shown to improve the heterologous expression of membrane-boundP450s in bacterial cells (Alkier, B. A. et al. Arch. Biochem. Biophys.322, 369-377 (1995), Haudenschield, et al Arch. Biochem. Biophys. 379,127-136 (2000)). In addition, for the entire cDNA, the codon usage wasadapted to match the E. coli codon usage. Thus, several cDNA weredesigned for CYP71AV8 with different 3′-end modifications andoptimizations:

-   -   CYP71AV8-65188: in this construct the 22 first codons were        replaced by a sequence coding for the MALLLAVFWSALIILV peptide        (SEQ ID NO 3 and 4).    -   CYP71AV8-P2: the entire anchor-encoding sequence was replaced by        the anchor sequence of an optimized limonene-hydroxylase from        mint (PM2 in Haudenschield, et al Arch. Biochem. Biophys. 379,        127-136 (2000)) (SEQ ID NO 5 and 6).    -   CYP71AV8-P2O: this construct encodes for the same protein as the        previous one but the membrane anchor region was further codon        optimize (SEQ ID NO 7 and 8).        The FIG. 1 compares the amino acid sequences of the N-terminal        regions of the different CYP71AV8 variants and FIG. 2 compares        the DNA sequences of the 3 constructs. The three optimized        CYP71AV8 cDNAs were synthesized in-vitro (DNA2.0, Menlo Park,        Calif., USA) and cloned as NdeI-HindIII fragment into the        pCWori+ expression plasmid (Barnes, H. J. Method Enzymol. 272,        3-14; (1996)).

Example 2 Functional Expression of CYP71AV8 in Bacterial Cells

For heterologous expression, the JM109 E. coli cells were transformedwith the CYP71AV8 expression plasmids (example 1). Single colonies oftransformants were used to inoculated cultures of 5 mL LB mediumcontaining 50 μg/mL ampicillin. The cells are grown for 10 to 12 hoursat 37° C. The cultures were then used to inoculate 250 mL TB Medium(Terrific Broth) supplemented with 50 μg/mL ampicillin and 1 mM ThiamineHCL. The cultures were incubated at 28° C. for 3-4 h with moderateshaking (200 rpm) before 75 mg/L δ-aminolevulinic acid (sigma) and 1 mMIPTG (Isopropyl β-D-1-thiogalactopyranoside) was added, and the cultureswere maintained at 28° C. for 24-48 h with 200 rpm shaking.

The expression of the P450 enzymes can be evaluated qualitatively andquantitatively by measuring the CO-binding spectrum (Omura, T. & Sato,R. (1964) J. Biol. Chem. 239, 2379-2387) in the E. coli proteinfractions. For protein extraction, the cells are centrifuged (10 min,5000 g, 4° C.) and resuspended in 35 mL ice-cold buffer 1 (100 mMTris-HCl pH 7.5, 20% glycerol, 0.5 mM EDTA). One volume of 0.3 mg/mllysozyme (Sigma-Aldrich) in water was added and the suspension left10-15 min at 4° C. with agitation. The suspension is centrifuged 10 minat 7000 g and 4° C. and the pellet is resuspended in 20 mL buffer 2 (25mM KPO₄ pH 7.4, 0.1 mM EDTA, 0.1 mM DTT, 20% glycerol). The suspensionis subject to one cycle of freeze-thaw at −80° C., 0.5 mM PMSF(phenylmethylsulfonyl fluoride, Sigma-Aldrich) is added and thesuspension is sonicated 3 times for 20 sec. The suspension iscentrifuged 10 min at 10000 g (to remove cell debris) and thesupernatant is recovered and centrifuged 2 hours at 100,000 g. Thepellet (membrane protein fraction) is resuspended in 2-3 ml of buffer 3(50 mM Tris-HCl pH 7.4, 1 mM EDTA, 20% glycerol). To measure theCO-spectrum, the protein fraction is diluted (1/10) in buffer 3 to afinal volume of 2 mL. Some crystals of sodium dithionite (Na₂S₂O₄) areadded, the sample is divided into two cuvettes and the baseline recordedbetween 370 and 500 nm. The sample cuvette is then saturated with carbonmonoxide and the difference spectrum is recorded. The concentration ofP450 enzyme can be estimated from the amplitude of the peak at 450 nmusing the extension coefficient for the reduced CO complex of 91mM⁻¹•cm⁻¹ (Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 2379-2387).

Following this procedure, typical CO-spectra with a maximum absorbanceat 450 nm were measured for the recombinant CYP71AV8, attesting for aproper folding into functional P450 enzymes.

Example 3 Co-Expression of CYP71AV8 and a P450-Reductase in Bacteria

To reconstitute the activity of plant P450s, the presence of a secondmembrane protein is essential. This protein, the P450-reductase (CPR),is involved in the transfer of electrons from the cofactor NADPH(reduced Nicotinamide adenine dinucleotide phosphate) to the P450 activesite. It has been shown that a CPR from one plant can complement theactivity of P450 enzyme from another plant (Jensen and Moller (2010)Phytochemsitry 71, 132-141). Several CPR-encoding DNA sequences havebeen reported from different plant sources. We first selected a CPRpreviously isolated from Mentha piperita (CPRm, unpublished data, SEQ IDNO 10), optimized the codon usage of the full-length cDNA (SEQ ID No 9)and cloned it into the NcoI and HindIII restriction sites of thepACYCDuet-1 expression plasmid (Novagen) providing the plasmidpACYC-CPRm.

CYP71AV8 and CPRm were co-expressed in E. coli cells using the twoplasmids pCWori-CYP71AV8-65188 and pACYCDuet-CPRm. BL21 Star™(DE3) E.coli cells (Invitrogen, Carlsbad, Calif.) were co-transformed with thesetwo plasmids. Transformed cells were selected on carbenicillin (50μg/ml) and chloramphenicol (34 μg/ml) LB-agarose plates. Single colonieswere used to inoculate 5 mL liquid LB medium supplemented with the sameantibiotics. The culture was incubated overnight at 37° C. The next day,2 to 250 mL of TB medium supplemented with the same antibiotics wereinoculated with 0.2 to 2 mL of the overnight culture. After 6 hoursincubation at 37° C., the culture was cooled down to 28° C. and 1 mMIPTG and 75 mg/L δ-aminolevulinic acid were added. After 16 to 24 hours,the cells were harvested in exponential growing phase, centrifuged andresuspended in 0.5 volume of potassium phosphate buffer 50 mM pH 7.0supplemented with 5% glycerol or 3% glucose. These cells were used forevaluation of the enzymatic activities of the P450 enzymes.

Example 4 Bioconversion of (+)-α-Santalene, (−)-β-Santalene,(−)-α-Trans-Bergamotene and (+)-Epi-β-Santalene Using E. coli CellsExpressing CYP71AV8

The different sequiterpene hydrocarbons used as substrates in thebioconversion assays were prepared as described previously using E. colicells engineered to produced farnesyl diphosphate (FPP) from anheterologous mevalonate pathway and expressing a plant derivedsesquiterpene synthase. The engineering and use of the E. coli hostcells was described in patent WO2013064411 or in Schalk et al (2013) J.Am. Chem. Soc. 134, 18900-18903. Briefly, an expression plasmid wasprepared containing two operons composed of the genes encoding theenzymes for a complete mevalonate pathway. A first synthetic operonconsisting of an E. coli acetoacetyl-CoA thiolase (atoB), aStaphylococcus aureus HMG-CoA synthase (mvaS), a Staphylococcus aureusHMG-CoA reductase (mvaA) and a Saccharomyces cerevisiae FPP synthase(ERG20) genes was synthetized in-vitro (DNA2.0, Menlo Park, Calif., USA)and ligated into the NcoI-BamHI digested pACYCDuet-1 vector (Invitrogen)yielding pACYC-29258. A second operon containing a mevalonate kinase(MvaK1), a phosphomevalonate kinase (MvaK2), a mevalonate diphosphatedecarboxylase (MvaD), and an isopentenyl diphosphate isomerase (idi) wasamplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334)and ligated into the second multicloning site of pACYC-29258 providingthe plasmid pACYC-29258-4506. This plasmid thus contains the genesencoding all enzymes of the biosynthetic pathway leading fromacetyl-coenzyme A to FPP. E. coli cells (BL21 Star™(DE3), Invitrogen)were co-transformed with the plasmid pACYC-29258-4506 and either theplasmid pET101-Cont2_1 (containing a cDNA encoding for the Clausenalansium (+)-α-santalene synthase (ClASS), WO2009109597) or the plasmidpETDuet-SCH10-Tps8201-opt (containing a cDNA encoding for a Santalumalbum (+)-α-santalene/(−)-β-santalene synthase (SaSAS), WO2010067309)and this cells were used to produce and purify (+)-α-santalene or amixture of (+)-α-santalene, (−)-β-santalene, (−)-α-trans-bergamotene and(+)-epi-β-santalene.

The enzymatic activity of CYP71AV8 was evaluated by bioconversion in E.coli cells using the sesquiterpene molecules listed above as substrates.BL21 Star™(DE3) E. coli cells (Invitrogen) transformed with the plasmidspACYCDuet-CPRm and pCWori-CYP71AV8-65188 were cultivated and harvestedas described in example 3. The substrates (sesquiterpene hydrocarbons)were added to the cell suspension to a final concentration of 0.5 mg/mlas mixture composed of 10 mg Tween® 20 (sigma-Aldrich), 10 mg antifoam(Erol DF, PMC Ouvrie, Lesquin, France), 20 mg sesquiterpene and 1 mlwater. The conversion was allowed to proceed for 24 hours at 20° C. withmoderate shaking. The media were extracted with 2 volumes of MTBE(Methyl tert-buthyl ether, Sigma) and the extracts were analyzed by GCMSon an Agilent 6890 Series GC system connected to an Agilent 5975 massdetector. The GC was equipped with 0.25 mm inner diameter by 30 m SPB-1capillary column (Supelco, Bellefonte, Pa.). The carrier gas was He at aconstant flow of 1 mL/min. The initial oven temperature was 80° C. (1min hold) followed by a gradient of 10° C./min to 300° C. Theidentification of the products was based on the comparison of the massspectra and retention indices with authentic standards and internaldatabases.

In these conditions, oxidation of (+)-α-santalene was observed. Theprimary product of the conversion was (E)-α-santalol. Other productsderived from the conversion of (E)-α-santalol by E. coli endogenousenzymes were detected: (E)-α-santalal (produced by an alcoholdehydrogenase) and (E)-α-dihydrosantalol (produced by an enoatereductase) (FIG. 3A). Similarly, using a mixture of (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene and (+)-epi-β-santalene assubstrate the formation of (E)-α-santalol, (E)-β-santalol,(E)-α-trans-bergamotol and (E)-epi-β-santalol was observed as well asfurther metabolized products were obtained (FIG. 3B). This example showsthat CYP71AV8 can be used for the terminal oxidation of (+)-α-santalene,(−)-β-santalene, and structurally similar molecules.

Example 5 Construction of Synthetic Operons to Co-Express CYP71AV8 and aCPR from a Single Plasmid

Several bicistronic operons were designed to express the P450 enzyme anda CPR from a single plasmid and under the control of a unique promoter.The three variants of optimized CYP71AV8 cDNAs (example 1) were combinedwith 2 CPR cDNAs: the codon optimized CPRm cDNA (example 2) and a codonoptimized cDNA (Seq ID No 11) encoding for an Artemisia annua CPR (NCBIaccession No. ABM88789.1, SEQ ID No 12). Thus, six constructs weredesigned (Seq ID No 13-18), each containing a P450 cDNA followed by alinker sequence including a ribosome binding site (RBS) and a CPR cDNA(FIG. 4). This constructs were prepared by PCR: the P450 and CPR cDNAswere amplified separately and with 5′ and 3′ overhangs suitable for thecloning using the In-Fusion® procedure (Clontech) in the NdeI-HindIIIsites of the pCWori+ plasmid.

To evaluate the effect of the different N-terminal modification made onthe P450s and the coupling with the CPRs, the 6 plasmids weretransferred into E. coli BL21 Star™(DE3) cells and the recombinant cellswere used in bio-conversion assays as described in example 4. The(+)-α-santalene and the (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene mixture were used assubstrates and quantities of total oxygenated sesquiterpene productswere evaluated. The results presented in FIG. 5 show that allrecombinant bacterial cells transformed with one of the 6 plasmidsdescribed above can be used for the oxidation of (+)-α-santalene,(−)-β-santalene and the structurally similar molecules. The highesttiter was obtained with the operon combining the CYP71AV8-P2O cDNA andthe CPRm cDNA. This construct (plasmid pCWori-CYP71AV8-P2O-CPRm) wasused for further experiments.

Example 6 In-Vivo Production of Oxygenated Sesquiterpenes in EngineeredCells

The oxidized products of (+)-α-santalene and the (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene, (+)-epi-β-santalene or otherstructurally similar molecules can also be produced directly in E. colicells engineered to produce sesquiterpenes from a carbon source such asglucose or glycerol. Plasmids were prepared consisting of the pCWori+plasmid (Barnes H. J (1996) Method Enzymol. 272, 3-14) containing asynthetic operon composed of a P450, a CPR and the terpene synthase. Forthe P450, the CYP71AV8-P2 or CYP71AV8-P2O cDNA was used and for theterpene synthase, the Clausena lansium (+)-α-santalene synthase cDNA(ClASS) (WO2009109597) or a cDNA encoding for a Santalum album(+)-α-santalene/(−)-β-santalene synthase (SaSAS) (WO2010067309) wasused. Four plasmids were thus constructed using the following procedure.A codon optimized version of the ClASS cDNA (SEQ ID NO 19-20) wasdesigned and synthesized (DNA 2.0) and cloned in the NdeI-KpnI sites ofthe pETDUET-1 plasmid (Novagen) providing the plasmid pETDuet-Tps2opt.For SaSAS an optimized full-length cDNA was designed (SEQ ID NO 21-22),synthesized and cloned in the pJexpress414 plasmid (DNA2.0) providingthe plasmid pJ414-SaTps8201-1-FLopt. For each constructs primer weredesigned for cloning using the In-Fusion® technique (Clontech, TakaraBio Europe). The optimized ClASS cDNA and the optimized SaSAS cDNA wereamplified using these primers and the pETDuet-Tps2opt andpJ414-SaTps8201-1-FLopt plasmids as template, respectively. The two PCRproducts were ligated in the plasmids pCWori-CYP71AV8-P2-CPRm orpCWori-CYP71AV8-P2O-CPRm digested with the HindIII restriction enzymeand using the In-Fusion® Dry-Down PCR Cloning Kit (Clontech, Takara BioEurope), providing four new plasmids: pCWori-CYP71AV8-P2-CPRm-ClASS,pCWori-CYP71AV8-P2-CPRm-SaSAS, pCWori-CYP71AV8-P2O-CPRm-ClASS, andpCWori-CYP71AV8-P2O-CPRm-SaSAS (SEQ ID NO 23-26).

The evaluation of the performance of these operons was performed in theE. coli BL21 Star™(DE3) (Invitrogen) cells co-transformed with either ofthe 4 plasmids and with the plasmid pACYC-29258-4506 carrying a completemevalonate pathway (example 4). Transformed cells were selected oncarbenicillin (50 μg/ml) and chloramphenicol (34 μg/ml) LB-agaroseplates. Single colonies were used to inoculate 5 mL of LB mediumsupplemented with appropriate antibiotics. Cultures were incubatedovernight at 37° C. and 250 rpm. The next day 2 mL of TB medium in glassculture tubes containing 100 μg/L carbenicilin and 17 μg/Lchloramphenicol, were inoculated with 200 μl of the LB pre-culture andincubated at 37° C. and 250 rpm. After 6 hours of cultivation (or whenthe optical density at 600 nm of the culture reach a value of 3), theculture were cooled down to 20° C. and the expression of the proteinswas induced with 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside),and 75 μg/L δ-aminolevulinic acid (sigma) and 2% (v/v) of decane wereadded. After 48 h incubation with 250 rpm shaking, the whole culturebroth was extracted with 1 volume of MTBE and analyzed by GCMS asdescribed in example 4.

All resulting strains produced the sesquiterpene hydrocarbons as well asthe corresponding oxygenated products also observed in the bioconversionexperiments (FIG. 6). This experiment shows that using engineered cellsexpressing CYP71AV8, the sesquiterpenes (E)-α-santalol, (E)-β-santaloland other structurally similar molecules can be produced.

Example 7 Production of (E)-α-Santalol and (E)-β-Santalol Using CYP71AV8Variants

In previous examples we showed that CYP71AV8 is highly selective for the‘terminal trans carbon’ of (+)-α-santalene and (−)-β-santalene andproduced exclusively (E)-α-santalol, (E)-β-santalol. In this example, wedescribe a site directed mutagenesis approach to modify the CYP71AV8enzyme activity in order to produce (Z)-α-santalol and (Z)-β-santalol.L358 was first selected as an active site residue controlling the enzymeactivity. A series of variant of CYP71AV8 were generated by replacingthe codon encoding for L358 by codons encoding for other amino acids.The mutation was introduced in a two-step PCR procedure using acombination of degenerated oligonucleotide (containing the NBT(N=A,C,G,T; B=C,G,T) codon in place of L358 encoding codon) and specificoligonucleotides. This combination of oligonucleotides allow to changethe L358 encoding codon with codons encoding for 12 other residuesincluding all the amino acids with a hydrophobic side chain. A first PCRwas performed to amplify the 5′ portion of the cDNA using themutagenesis reverse primer AV8-L358-rev(5′-CACGCGGCATCACCAGCGGAVNCGGCGGATGCAGGCGCAGGGTTTCTTTAATC-3′) (SEQ IDNO: 93) and the primer AV8-pcw-fw(5′-CATCGATGCTTAGGAGGTCATATGGCTCTGTTATTAGCAG-3′) (SEQ ID NO: 94). Asecond PCR product was amplified using the primer AV8-L358-fw(5′-TCCGCTGGTGATGCCGCGTGAGTGC-3′) (SEQ ID NO: 95) and AV8-CPR-rev(5′-ATATATCTCCTTCTTAAAGTTAGTCGACTCATTAGGTG-3′) (SEQ ID NO: 96). For bothamplifications the pCWori-CYP71AV8-P2-CPRm-ClASS was use for thetemplate. A second round of amplification was performed using the twoabove PCR products as template and the primers AV8-L358-fw+AV8-CPR-revand allowed to amplify the full-length CYP71AV8 variant cDNAs. All thePCR reactions were performed using the PfuUltra II fusion HS DNApolymerase (Stratagene) following the manufacturer instruction. Themodified cDNA were ligated into the NdeI-SalI digestedpCWori-CYP71AV8-P2-CPRm-ClASS using the Gibson Assembly Master Mix (NewEngland Biolabs). The final constructions were controlled by sequencingand one plasmid clone was selected for each desired CYP71AV8 variant.Twelve variants were thus generated by replacing Leu358 by Ala, Phe,Thr, Ser, Val, Gly, Ile, Met, Pro, Tyr, Trp, and Arg (SEQ ID NO 27 to50).

The evaluation of each CYP71AV8 variant was performed using the in-vivosesquiterpene production method described in example 6. Briefly, thepCWori+ plasmid containing one of the CYP71AV8 variant cDNA, the CPRmcDNA and the ClASS cDNA was co-transformed with the pACYC-29258 plasmidinto KRX E. coli cells (Promega). The transformed cells were selected,cultivated and the production of sesquiterpenes was evaluated asdescribed in example 6. As shown in FIG. 7, compared to the wild typeP450 enzyme, with some of the variants (Z)-α-santalol was produced inaddition to the trans oxidation products. For each variant, the ratio ofcis to trans oxidation was calculated by dividing the total amount of(Z)-α-santalol produced by the total amount of oxygenated α-santalenederivatives. The results of these calculations for each variants ispresented in Table 1 below:

TABLE 1 Regio-selectivity of the CYP71AV8 wild-type enzyme and activesite variants for the oxidation of α-santalene. Titer [mg/L] %(Z)-α-santalol CYp71AV8 Oxygenated of total santalol variantssesquiterpenes products content CYP71AV8 wt 97.7 ± 2.8 78.1 ± 3.5 0%L358A  28 ± 1.5 40.3 ± 2.8 36%  L358F 88.4 ± 3.9 40.7 ± 0.4 46%  L358T90.9 ± 5.6 33.8 ± 1.5 5% L358S 43.4 ± 1.8 15.3 ± 0.8 17%  L358V 56.1 ±3.5 66.1 ± 1.1 1% L358G 84.3 ± 2.8  85 ± 2.2 0% L358I 71.2 ± 3.7  41 ±0.3 0% L358M  84 ± 4.5  2.3 ± 0.3 0% L358P 71.6 ± 2.0  21 ± 1.1 0% L358Y71.6 ± 2.9  0 ± 0 0% L358W 78.2 ± 0.6  0 ± 0 0% L358R  76 ± 1.1  2.3 ±0.3 0%

The data presented in Table 1 above show that CYP71AV8 can be engineeredand used to produce the (Z)-α-santalol. Particularly, the L358T, L358S,L358A and L358F variants can be used for the terminal oxidation of(+)-α-santalene with a selectivity up to 46% for the cis terminalcarbon.

In a similar approach the variants of CYP71AV8 were evaluated for theproduction of (Z)-β-santalol. New plasmids were prepared by replacingthe ClASS cDNA in the above plasmid by the SaSAS cDNA. Thus the plasmidpCW-CYP71AV8-L358F-CPRm-ClASS was digested with the restriction enzymesHindIII and EcoRI to remove the ClASS cDNA. In parallel, thepCWori-CYP71AV8-P2-CPRm-SaSAS was digested with the same enzymes torecover the SaSAS cDNA with the compatible cohesive ends. The linearizedvector and the digested insert were ligated using the T4 DNA ligase (NewEngland Biolabs). The plasmid thus obtained was used for in-vivoproduction of oxygenated sesquiterpenes in E. coli cells in the samecondition as described above. The FIG. 8 present the GCMS profile of theanalysis of the products formed by CYP71AV8-L358F and shows thatmodified CYP71AV8 enzymes can also be used to produce (Z)-β-santalol.

Example 8 Evaluation of Other Members of CYP71AV Family

CYP71AV1 (NCBI accession No ABB82944.1) was evaluated for the oxidationof sesquiterpenes with the santalene skeleton. A plasmid was preparedwith a configuration similar to the plasmids described in example 5: abi-cistronic operon containing an optimized cDNA encoding for anN-terminal modified CYP71AV1 protein (SEQ ID NO 53 and 54) and the aaCPRcDNA (example 5) was designed, synthesized in-vitro (DNA2.0) and clonedas a bi-cistronic operon into the pCWori+ plasmid. The plasmid was usedto transform KRX E. coli cells (Promega). The transformed cells werecultivated and protein expression was induced as described in example 3.A bioconversion experiment using (+)-α-santalene as substrate wasconducted as described in example 4. As shown in FIG. 9 the sameproducts as with CYP71AV8 were obtained (i.e. (E)-α-santalol and(E)-α-santalal) showing that other members of the CYP71AV P450 familycan be use for the terminal oxidation of santalenes.

Using CYP71AV1, a synthetic operon containing the CYP71AV1 cDNA, theaaCPR and the (+)-α-santalene synthase cDNA (ClASS) was prepared. ThepCWori+ plasmid containing the CYP71AV8-P2-CPRm-ClASS operon (example 6)was digested with NdeI and HindIII to cut out the P450 encoding cDNA. Inparallel, the CYP71AV1 cDNA was recovered from the bi-cistronic operondescribed in the previous paragraph by digestion with the same enzymesand ligated, using the T4 DNA ligase (New England Biolabs), into thedigested pCWori plasmid described above yielding the plasmidpCWori-CYP71AV1-CPRm-ClASS. This plasmid together with the plasmidpACYC-29258-4506 were used to co-transform E. coli BL21 Star™(DE3)(Invitrogen) cells. The recombinant cells were cultivated in conditionsallowing the production of sesquiterpene molecules as described inexample 6. The GCMS analysis of the sesquiterpene produced revealed theformation of the same product as in the bio-conversion experiments. Thisexperiment shows that CYP71AV1 can also be used oxidize santalenemolecules and to produce santalols (FIG. 9).

Example 9 Construction of a P450-BM3 (CYP102A1) Mutant Library

A P450-BM3 mutant library of 24 variants was constructed bysystematically combining five hydrophobic amino-acids (alanine, valine,phenylalanine, leucine and isoleucine) in two positions located close tothe centre of the heme group of P450-BM3. Altering the side chain sizeof these two amino acids has been shown to drastically change the shapeof the substrate binding cavity in close proximity of the heme group(Appl Microbiol Biotechnol 2006, 70:53; Adv Synth Catal 2006, 348:763).The first hot spot (Phe 87) is known to alter substrate specificity andregioselectivity while the second position (Ala 328) has been predictedto interact with all substrates during oxidation (ChemBiochem 2009,10:853). The P450-BM3 variants were either generated using theQuickChange™ site-directed mutagenesis kit (Invitrogen, Carlsbad,Calif.) or were chemically synthetized by DNA2.0 (Menlo Park, Calif.).The P450-BM3 variants and wild-type were subcloned into the bacteriaexpression plasmids pET22b, pET28+, pETDuet-1 and pCDFDuet-1 (Novagen,Madison, Wis.) and were transformed in Escherichia coli BL21(DE3) orBL21Star™(DE3) (Invitrogen, Carlsbad, Calif.).

Example 10 Alpha-Santalene: In Vitro Screening of the P450-BM3 Library

The 24 P450-BM3 mutants and the wild-type version of the enzyme wereheterologously expressed in E. coli BL21(DE3) cells as reportedpreviously (Adv. Synth. Catal. 2003, 345:802). In brief, a single colonyof transformed cells was used to inoculate 2 ml of Luria-Bertani (LB)medium supplemented with 30 μg/ml kanamycin and grown at 37° C. withorbital shaking (150 rpm) until OD₅₇₈ reaches a value of 0.6 to 1.0.This pre-culture was used to inoculate 200 ml of LB medium containing 30μg/ml kanamycin. The cells were grown at 37° C. with orbital shaking at160 rpm to an OD₅₇₈ of 0.8. Expression of the protein was then inducedby the addition of 0.35 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).After 6 hours of growth at 30° C. under agitation, the cells wereharvested by centrifugation and lysed by sonication.

The alpha-santalene used as substrate in the bioconversion assays wasprepared as described in Example 4. The conversions were carried out in1 ml of 50 mM potassium phosphate buffer containing ˜0.5 μM CYP enzyme,2% (v/v) DMSO, and 0.2 mM μ-santalene substrate. Reaction was started byadding 0.1 mM NADPH and was carried out for 22 h at room temperaturewith moderate shaking.

Samples were then analyzed on a GC/MS QP-2010 instrument (Shimadzu,Japan) equipped with a FS-Supreme-5 column (30 m×0.25 mm×0.25 μm),helium as carrier gas (flow rate: 0.68 ml/min; linear velocity: 30cm/s). Mass spectra were collected using electrospray ionization. Theinjector temperature was set at 250° C. The column oven was set at 50°C. for 1 min, then raised to 170° C. at 30° C./min, then raised to 185°C. at 5° C./min, held isotherm for 3 min, then raised to 200° C. at 5°C./min, then raised to 300° C. at 30° C./min, and finally held isothermfor 1 min.

Example 11 Alpha-Santalene In Vivo Screening of the P450-BM3 Library

The P450-BM3 mutant library was also screened in vivo using a bacteriastrain engineered to produce (+)-α-santalene from a simple carbonsource. To this end, the FPP-overproducing strain described in Example 4was transformed with a pETDuet-1 plasmid containing a codon-optimizedversion of a (+)-α-santalene synthase from Clausena lansium (ClASS)(WO2009109597) (SEQ ID No 19 and 20) and each of the P450-BM3 variantscloned into the first and second multiple cloning sites (MCS) of thevector, respectively. Alternatively, the (+)-α-santalene synthase cDNAwas cloned into the pET101expression plasmid (Novagen) and each of theP450-BM3 mutants from the library into the pCDFDuet-1 vector (Novagen).The resulting recombinant vectors were co-transformed in theFPP-overproducing strain.

Single colonies of transformed cells were used to inoculate 5 mL of LBmedium supplemented with the appropriate antibiotics. Cultures were thenincubated overnight at 37° C. and 250 rpm. The following day, 2 mL ofTerrific Broth (TB) medium supplemented with 3% glycerol, 1 mMthiamine-HCl (Sigma-Aldrich, St Louis, Mich.) and 75 μg/Lδ-aminolevulinic acid (Sigma-Aldrich) were inoculated with 200 μl of theovernight culture and incubated at 37° C. and 250 rpm. After 4 to 6hours of cultivation (or when the optical density at 600 nm of theculture reach a value of 2 to 3), the cultures were cooled down to 28°C. and the protein expression was induced with 0.1 mM IPTG.

At that time, 10% (v/v) of dodecane were added to the growth media.After 48 h incubation with orbital shaking (250 rpm), the cell culturewas extracted twice with one volume of methyl tert-butyl ether (MTBE)and the solvent extract analyzed by GC/MS. GC/MS was performed on anAgilent 6890 series GC system equipped with a DB1 column (30 m×0.25mm×0.25 mm film thickness; Agilent) and coupled with a 5975 series massspectrometer. The carrier gas was helium at a constant flow of 1 ml/min.Injection was in split-less mode with the injector temperature set at250° C. and the oven temperature was programmed from 50° C. to 225° C.at 10° C./min and to 320° C. at 20° C./min. The identities of theproducts were confirmed based on the concordance of the retentionindices and mass spectra of authentic standards.

The in vitro (Example 10) and in vivo screening of the P450-BM3 mutantlibrary gave comparable results that are summarized in Table 2. WhileP450-BM3 wild-type (SEQ ID No 55 and 56) did not show any detectableactivity on (+)-α-santalene, 6 P450-BM3 variants were able to convertα-santalene to the desired α-santalol(s). These variants revealedbetween 45% to 96% preference for oxidation of the cis-terminal carbonof (+)-α-santalene. The single mutant #23 (A328V) (SEQ ID No 67 and 68)and the double mutants #7 (F87I/A328I) (SEQ ID No 57 and 58), #17(F87V/A328I) (SEQ ID No 59 and 60) and #18 (F87V/A328L) (SEQ ID No 61and 62) showed the highest regioselectivity ranging from 72% to 96%(Table 2 and FIG. 10). Two additional variants #19 (F87V/A328V) (SEQ IDNo 63 and 64) and #20 (F87V/A328F) (SEQ ID No 65 and 66) were lessselective for the cis-hydroxylation (in the range of 45%-50%) andgenerated additional oxidation products.

TABLE 2 Alpha-santalene conversion to alpha- santalol(s) by P450-BM3variants Additional cis-α- trans-α- oxydation Conver- santalol santalolproducts sion P450-BM3 (%) (%) (%) (%) Wild-type F87/A328 Variant #7 F87I/A328 I 87 13 6 Variant #17 F87 V/A328 I 78 11 11 16 Variant #18 F87V/A328 L 72 13 15 9 Variant #19 F87 V/A328 V 45.5 4 50.5 8 Variant #20F87 V/A328 F 49 8 43 3 Variant #23 F87/A328 V 96 4 5

These results indicate that P450-BM3 active site mutations enablebinding of the non-native substrate (+)-α-santalene. Selected P450-BM3variants incorporating these mutations were shown to selectivelyhydroxylate the cis-terminal carbon of (+)-α-santalene to generate theolfactively important compound (Z)-α-santalol (FIG. 10).

Example 12 In Vivo Production of (Z)-α-Santalol, (Z)-β-Santalol,(Z)-α-Trans-Bergamotol and (Z)-Epi-β-Santalol Using a P450-BM3 DoubleMutant

One of the P450-BM3 variants identified in the α-santalene screen(variant #17; Table 2) was tested for its ability to oxidize asandalwood oil-like mixture of sesquiterpene hydrocarbons consisting of(+)-α-santalene, (−)-β-santalene, (−)-α-trans-bergamotene and(+)-epi-β-santalene. To this end, the FPP-overproducing bacteria straindescribed in Example 4 was transformed with a recombinant pETDuet-1expression vector containing a codon-optimized cDNA encoding for aSantalum album (+)-α-santalene/(−)-β-santalene synthase (WO2010067309)(SEQ ID No 21 and 22) into the first MCS and the P450-BM3 variant #17 inthe second MCS. Cell growth, induction conditions, culture extractionand product analysis were performed essentially as described in Example11.

As shown in FIG. 11, (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene were efficientlyoxidized by the P450-BM3 double-mutant to yield (Z)-α-santalol,(Z)-β-santalol, (Z)-α-trans-bergamotol and (Z)-epi-β-santalol.Remarkably, only the desired cis-isomers of the sesquiterpene alcoholswere detected under these experimental conditions. These data show thatthe Bacillus megaterium CYP102A1 (P450-BM3) can be efficiently engineerto selectively hydroxylate the cis-terminal carbon of (+)-α-santalene,(−)-β-santalene and structurally related terpenes such as bergamotanesesquiterpenes and to generate the key sesquiterpene alcohols found inSandalwood oil.

Example 13 Isolation of a cDNA Encoding for SaCP816, a Cytochrome P450from Santalum album

The seeds of S. album were obtained from B&T World Seeds (Aigues-Vives,France) and from Sandeman Seeds (Lalongue, France). The seeds were firstsurface sterilised in 2.5% Hypochlorous acid (HClO) for 120 min, andrinsed 3 times in sterile ultrapure water. The seeds were then shelledand placed on MS basal medium (Murashige & Skoog, 1962, PhysiologiaPlantarum 15, 473-497) supplemented with 15 g/L sucrose and 7.8 g/Lagar, pH 5.7. Germination was typically observed after 9 to 18 days witha yield of approximately 40%. Seedlings of Santalum album obtained fromthe aseptically germinated seeds were transferred to soil 5 to 10 weeksafter germination. Since Santalum species are root hemiparasites, thesoil adaptation was made in close contact with 6-months to 1-year oldcitrus (Citrus sinensis) plants. The roots of the Santalum plants wereharvested, 2-3 years after the transfer to the soils and separated fromthe host plant roots. GC-MS analysis of an extract of these roots showedthe presence of the sandalwood oil characteristic sesquiterpenes. TotalRNA was extracted from the roots using the Concert™ Plant RNA Reagent(Invitrogen). From 12 grams of tissue, 640 micrograms of total RNA wereisolated.

The whole transcriptome was sequenced using the Illumina Total RNA-Seqtechnique and the Illumina HiSeq 2000 sequencer. A total of 108.7millions of paired-reads of 2×100 bp were generated. The reads wereassembled using the De Novo Assembly application of CLC-Bio GenomicWorkbench (CLCBo, Denmark). A total 82'479 of contigs with an averagesize of 683 bp were assembled. The contigs were search using the tBlastnalgorithm (Altschul et al, J. Mol. Biol. 215, 403-410, 1990) and usingas query sequence known P450 amino acid sequences such as the sequenceof CYP71AV1 (NCBI accession No ABB82944.1). This approach allowedidentifying several contigs encoding for proteins with characteristiccytochrome P450 motifs. One selected contig, SCH37-Ct816 (SEQ ID NO 69),contained a 1503 bp length open reading frame (ORF) (SEQ ID NO 70)encoding for a 500 amino acid protein, SaCP816 (SEQ ID NO 71). Thisamino acid showed homology with know cytochrome P450 sequences theclosest sequence being a P450 from Vitis vinifera, CYP71D10 (NCBIaccession No AAB94588.1) sharing 62% amino acid sequence identity.

Example 14 Heterologous Expression of SaCP816 in Bacterial Cells

For functional characterization of the protein encoded by SCH37-Ct816,the protein was heterologously expressed in E. coli cells. The ORFsequence was modified for improved expression in E. coli: the first 17codons were replaced by the codons encoding for the MALLLAVFWSALIILVpeptide (first 17 amino acids of SEQ ID NO: 73) and the codon usage ofthe whole ORF sequence was modified to match the E. coli codon usage.This cDNA (SaCP120293 (SEQ ID NO: 72) encoding for the modified SaCP816(SEQ ID NO: 73) was synthesized in-vitro (DNA2.0) and cloned in thepJExpress404 plasmid (DNA2.0). The heterologous expression was performedas described in example 2.

Example 15 Co-Expression of SaCP816 and a P450-Reductase in Bacteria

A bicistronic operons was designed to express the P450 enzyme and a CPRfrom a single plasmid and under the control of a unique promoter. Theoptimized SaCP120293 cDNA was combined with the CPRm cDNA (SEQ ID No 9,Example 3) to prepare a bicistronic construct (SEQ ID NO 74) containingsuccessively the P450 cDNA a linker sequence including a ribosomebinding site (RBS) and the CPRm cDNA. This construct was prepared by PCRby amplifying the P450 and CPR cDNAs separately and with 5′ and 3′overhangs suitable for the cloning using the In-Fusion® procedure(Clotech) in the NdeI-HindIII sites of the pCWori+ plasmid (Barnes H. J(1996) Method Enzymol. 272, 3-14) providing the plasmidSaCP816-CPRm-pCWori (SEQ ID NO 74).

The JM109 E. coli cells were transformed with the SaCP816-CPRm-pCWoriexpression plasmid. The transformed cells were grown and the cell-freeextract containing the recombinant proteins were prepared as describedin example 2. This protein fraction was used for the evaluation theenzymatique conversion of sesquiterpene molecules (example 16).

Example 16 In-Vitro Conversion of (+)-α-Santalene, (−)-β-Santalene,(−)-α-Trans-Bergamotene and (+)-Epi-β-Santalene Using the RecombinantSaCP816 P450 Enzyme

The different sequiterpene hydrocarbons used as substrates in thebioconversion assays were prepared as described in example 4.

The crude protein extract from E. coli cells expressing the recombinantSaCP816 and CPRm proteins (example 15) was used for the in-vitrooxidation of these sesquiterpene molecules. The assays were performed in1 mL of 100 mM Tris-HCL pH 7.4 buffer containing 20 to 50 microL proteinextract, 500 microM NADPH (reduced Nicotinamide adenine dinucleotidephosphate), 5 microM FAD (Flavine adenine dinucleotide), 5 microM FMN(flavine mononucleotide), and 300 microM of sesquiterpenes (either(α)-santalene or a mixture of (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene). After 2 hours ofincubation in Teflon sealed glass tubes with gentle agitation, thereaction was stopped on ice and extraction with 1 volume of MTBE (Methyltert-buthyl ether, Sigma). The extracts were analyzed by GCMS asdescribed in example 4.

In these conditions, oxidation of (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene was observed. FIG. 12shows that the oxidation of (+)-α-santalene by SaCP816 provides(Z)-α-santalol. FIG. 13 shows that (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene were oxidized by SaCP816to forme (Z)-α-santalol, (Z)-β-santalol, (Z)-α-trans-bergamotol and(Z)-epi-β-santalol. In all assays, no detectable amounts of thecorresponding trans-isomers of the sesquiterpene alcohols was observed(the trans and cis isomers of each sesquiterpene alcohol are easilyseparated in the chromatographic conditions used in these assays).

This experiments show that the cytochrome P450 enzymes, SaCP816,isolated from Santalum album can be used for the selective hydroxylatesthe cis-terminal carbon of (+)-α-santalene, (−)-β-santalene and similarsesquiterpene structures.

Example 17 In-Vivo Production of Oxygenated Sesquiterpenes in EngineeredCells Using the Recombinant SaCP816 P450 Enzyme

The oxidized products of (+)-α-santalene and the (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene, (+)-epi-β-santalene or otherstructurally similar molecules can also be produced directly in E. colicells engineered to produce sesquiterpenes from a carbon source such asglucose or glycerol. Plasmids were prepared consisting of the pCWori+plasmid containing a synthetic operon composed of the SaCP120293 cDNA(SEQ ID No 72), the CPRm cDNA (SEQ ID No 9) and a terpene synthaseencoding cDNA. For the terpene synthase, the Clausena lansium(+)-α-santalene synthase cDNA (ClASS) (WO2009109597) or a cDNA encodingfor a Santalum album (+)-α-santalene/(−)-β-santalene synthase (SaSAS)(WO2010067309) was used.

Two plasmids were thus constructed using a procedure similar to theprocedure described in example 6. The codon optimized (+)-α-santalenesynthase cDNA (SEQ ID NO 19) and the (+)-α-santalene/(−)-β-santalenesynthase cDNA (SEQ ID NO 21) were amplified as described in example 6and ligated using the In-Fusion® Dry-Down PCR Cloning Kit (Clontech,Takara Bio Europe) in the plasmids SaCP816-CPRm-pCWori digested with theHindIII restriction enzyme providing the two new plasmidsSaCP816-CPRm-ClASS-pCWori (SEQ ID NO 75) and SaCP816-CPRm-SaSAS-pCWori(SEQ ID NO 76).

The evaluation of the performance of these operons was performed in theE. coli XRX cells (Promega) co-transformed with either of these 2plasmids and with the plasmid pACYC-29258-4506 carrying a completemevalonate pathway (example 4). Transformed cells were selected oncarbenicillin (50 μg/ml) and chloramphenicol (34 μg/ml) LB-agaroseplates. Single colonies were used to inoculate 5 mL of LB mediumsupplemented with appropriate antibiotics. Cultures were incubatedovernight at 37° C. and 250 rpm. The next day 2 mL of TB medium in glassculture tubes containing 100 μg/L carbenicilin and 17 μg/Lchloramphenicol, were inoculated with 200 μl of the LB pre-culture andincubated at 37° C. and 250 rpm. After 6 hours of cultivation (or whenthe optical density at 600 nm of the culture reach a value of 3), theculture were cooled down to 20° C. and the expression of the proteinswas induced with 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and0.1% Rhamnose, and 75 μg/L δ-aminolevulinic acid (sigma) and 2% (v/v) ofdecane were added. After 48 h incubation with 250 rpm shaking, the wholeculture broth was extracted with 1 volume of MTBE and analyzed by GCMSas described in example 4.

All resulting strains produced the sesquiterpene hydrocarbons as well asthe corresponding oxygenated products also observed in the in-vitroexperiments (FIG. 14). This experiment shows that using engineered cellsexpressing SaCP816, the sesquiterpenes (Z)-α-santalol, (Z)-β-santaloland other structurally similar molecules can be produced.

Example 18 Isolation of a cDNA Encoding SaCP10374, a Cytochrome P450from Santalum album

As described in example 13, several P450-encoding contig sequences wereidentified in the transcriptome from Santalum album roots. BesideSCH37-Ct816, another contig was selected: SCH37-Ct10374 (SEQ ID NO 77),contained a 1533 bp length ORF (SEQ ID NO 78) encoding for a proteincomposed of 510 amino acids, SaCP10374 (SEQ ID NO 79), showing homologywith know cytochrome P450 sequences and 58% identity with CYP71D10 fromVitis vinifera, CYP71D10.

Example 19 Heterologous Expression of SaCP10374 in Bacterial Cells andCo-Expression with a P450-Reductase in Bacteria

For functional characterization of the enzymes encoded by SCH37-Ct10374,the protein was heterologously expressed in E. coli cells. The ORFssequence were modified to improve the expression in E. coli: the 18first codons were replaced by the codons encoding for the MALLLAVFWSALIIpeptide and the codon usage of the whole ORF sequence was optimized. Thenew cDNA, SaCP120292 (SEQ ID NO 80), encoding for the modified SaCP10374(SEQ ID NO 81) was synthesized in-vitro (DNA2.0) and cloned in thepJExpress404 plasmid (DNA2.0).

The heterologous expression was performed as described in example 2.Following this procedure, typical CO-spectra with a maximum absorbanceat 450 nm was measured for this new recombinant S. album P450, attestingfor a proper folding into functional P450 enzymes.

To reconstitute the activity of this P450 enzyme, a P450 reductase (CPR)was coexpressed. For this purpose, a bicistronic operons was designedsimilarly as described in example 15 to express SaCP10374 and CPRm (amint P450 reductase) from a single plasmid and under the control of aunique promoter. The optimized SaCP12092 cDNA was combined with the CPRmcDNA to prepare the bicistronic constructs (SEQ ID NO 82) containingsuccessively the P450 cDNA a linker sequence including a ribosomebinding site (RBS) and the CPRm cDNA. This construct was prepared by PCRas described in example 15. and cloned in the pCWori+ plasmid (Barnes H.J (1996) Method Enzymol. 272, 3-14) providing the plasmidSaCP10374-CPRm-pCWori. The JM109 E. coli cells were transformed withthese bicistronic expression plasmid. The transformed cells were grownand the cell-free extract containing the recombinant proteins wereprepared as described in example 2. The membrane protein fractions wereused for the evaluation the enzymatique conversion of sesquiterpenemolecules (example 21)

Example 21 In-Vitro Conversion of (+)-α-Santalene, (−)-β-Santalene and(−)-α-Trans-Bergamotene Using the Recombinant SaCP10374 P450 Enzyme

The different sequiterpene hydrocarbons (either (α)-santalene or amixture of (+)-α-santalene, (−)-β-santalene, (−)-α-trans-bergamotene and(+)-epi-β-santalene) used as substrates in this example of bioconversionassays were prepared as described in example 4.

The crude protein extract from E. coli cells expressing the recombinantSaCP10374 and CPRm proteins (example 20) was used for the in-vitrooxidation of these sesquiterpene molecules and the assays were performedas described in example 16. After 2 hours of incubation in Teflon sealedglass tubes with gentle agitation, the reaction was stopped on ice andextraction with 1 volume of MTBE (Methyl tert-buthyl ether, Sigma). Theextracts were analyzed by GCMS as described in example 4.

In these conditions, oxidation of (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene by SaCP10374 wasobserved. FIGS. 15 and 16 show that (+)-α-santalene, (−)-β-santalene,(−)-α-trans-bergamotene and (+)-epi-β-santalene were oxidized bySaCP10374 to form (E)-α-santalol, (E)-β-santalol, (E)-α-trans-bergamotoland (E)-epi-β-santalol. In all assays, no detectable amounts of thecorresponding cis-isomers of the sesquiterpene alcohols was observed(the trans and cis isomers of each sesquiterpene alcohol are easilyseparated in the chromatographic conditions used in these assays).

This experiments show that the cytochrome P450 enzyme SaCP10374,isolated from Santalum album, can be used for the selectivehydroxylation of the trans-terminal carbon of (+)-α-santalene,(−)-β-santalene and structurally similar sesquiterpene molecules.

Example 22 In-Vitro Conversion of (E)-β-Farnesene, (E)-α-Farnesene,(−)-Sesquisabinene B, (−)-β-Bisabolene and (−)-α-Trans-Bergamotene Usingthe Recombinant SaCP816 and SaCP10374 P450s Enzyme

Using the method described in example 4, several sequiterpenehydrocarbons structurally similar to the santalenes were prepared. The(−)-sesquisabinene B and (−)-β-bisabolene were produced using thepETDuet expression plasmid containing either a cDNA encoding forSaTps647, a Santalum album (−)-sesquisabinene B synthase (NCBI accessionNo. ADP37190.1) or a cDNA encoding for SaTps30, a Santalum album(−)-β-bisabolene synthase (NCBI accession No. ADP37189.1), incombination with the pACYC-29258-4506 plasmid described in example 4.The β-farnesene was obtained from Bedoukian (Danbury, Conn., USA),α-farnesene was from Treatt (Suffolk, UK) and (−)-α-trans-bergamotenewas purified from citrus oil.

The crude protein extracts from E. coli cells expressing the recombinantSaCP816 or SaCP10374 together with CPRm proteins (example 15 and 20)were used for the in-vitro oxidation of these sesquiterpene molecules.The assays and product identification by GCMS analysis was performed asdescribed in example 16.

In these conditions oxidation of (E)-β-farnesene, (E)-α-farnesene,(−)-sesquisabinene B, (−)-β-bisabolene and (−)-α-trans-bergamotene, wasobserved (FIGS. 17 to 21). For all these compounds, the two S. albumP450s are regioselective for one of the two carbons of the terminalgem-dimethyl group (R1 or R2 in FIG. 27): SaCP816 catalyzes theselective oxidation of the carbon atom of the methyl in cis positionrelative to the terminal double bond (R1 in FIG. 27), whereas SaCP10374catalyzes the oxidation of the same substrates exclusively on the carbonatom of the methyl group in trans relative to the terminal double bond(R2 in FIG. 27). The trans and cis isomers of each sesquiterpene alcoholare easily separated in the chromatographic conditions used in theseassays. The formation of the corresponding aldehyde when thetrans-methyl group is oxidyzes is attributed to E. coli endogenousalcohol dehydrogenase activity.

This experiments show that the cytochrome P450 enzymes, SaCP816 andSaCP10374, isolated from Santalum album can be used for the selectivehydroxylation of the cis-terminal and trans-terminal carbon,respectively, of various sesquiterpene molecules have structuresimilarities with β-farnesene, α-farnesene, (+)-α-santalene,(−)-β-santalene, (−)-α-trans-bergamotene, (−)-sesquisabinene B or(−)-β-bisabolene.

Example 23 In-Vivo Production of Various Oxygenated Sesquiterpenes inEngineered Cells Using the Recombinant SaCP816 or SaCP10374 P450s Enzyme

The oxidized sesquiterpene molecules described in example 21 and 22 canalso be produced directly using whole cells, such as for example E. colicells engineered to produce sesquiterpenes from a carbon source such asglucose or glycerol. Plasmids were prepared consisting of the pCWori+plasmid containing a synthetic operon composed of the SaCP120293 cDNA(SEQ ID No 72), or the SaCP120292 (SEQ ID No 80), the CPRm cDNA (SEQ IDNo 9) and a terpene synthase encoding cDNA (encoding either for anArtemisia annua β-farnesene synthase cDNA (NCBI accession NoAAX39387.1.1), a Picea abies α-farnesene synthase (NCBI accession NoAAS47697.1), a S. album (−)-Sesquisabinene B (NCBI accession NoADP37190.1), a S. album (−)-β-Bisabolene synthase (NCBI accession NoADP37189.1), a Clausena lansium α-santalene synthase (NCBI accession NoADR71055.1) or a S. album α-/β-santalene synthase (NCBI accession NoADP30867.1)).

The plasmids carrying the different combinations of synthetic operonswere prepared using the following procedure. The plasmid pD444-SR-AaBFS(containing an optimized cDNA encoding for AaBFS, an Artemisia annua(E)-β-farnesene synthase (NCBI accession No AAX39387.1), the plasmidpD444-SR-PaAFS (containing an optimized cDNA encoding for PaAFS, a Piceaabies (E)-α-farnesene synthase (NCBI accession No. AAS47697.1) were usedto amplify by PCR the (E)-β-farnesene synthase and (E)-α-farnesenesynthase cDNAs, respectively. The plasmids pETDuet-SaTps647andpETDuet-SaTps30 (example 22) were used as template to amplify by PCR thesesquisabinene B synthase and the bisabolene synthase cDNAs,respectively. For each constructs primer were designed for the cloningusing the In-Fusion® technique (Clontech, Takara Bio Europe). The AaBFScDNA was amplified using the forward primer CPRm_aaBFS_Inf1(TTACCTGCGTGATGTGTGGTAATAAAAGCTTAGGAGGTAAAAATGTCTACCCTGCCAATTTCTTC) (SEQID NO: 97) and the reverse primer AaBFS_Inf2(ATGTTTGACAGCTTATCATCGATAAGCTGAATTCTTACACAACCATCGGGTGCACAAAGAATG) (SEQID NO: 98). The PaAFS cDNA was amplified using the forward primer CPRmPaAFS_Inf1(TTACCTGCGTGATGTGTGGTAATAAAAGCTTAGGAGGTAAAAATGGATCTGGCAGTGGAAATCGC) (SEQID NO: 99) and the reverse primer PaAFS_Inf2(CTCATGTTTGACAGCTTATCATCGATAAGCTGAATTCTTACATCGGGACCGGCTCCAGGACGGTGC)(SEQ ID NO: 100). The SaTps647 cDNA was amplified using the primerforward CPRm_Tps_647_inf1(5′GCGTGATGTGTGGTAATAAAAGCTTAGGAGGTAAAAATGGCGACCGTTGTGGATGATTCT-3′) (SEQID NO: 101) and the primer reverse Tps647_Inf2(GCTTATCATCGATAAGCTGAATTCTTACTCTTCATCCAGGGTAATCGGGTGG) (SEQ ID NO: 102).The SaTps30 cDNA was amplified using the primer forwardCPRm_Tps30_Inf1-(GCGTGATGTGTGGTAATAAAAGCTTAGGAGGTAAAAATGGACGCATTCGCAACGAGCC)(SEQ ID NO: 103) and the primer reverse Tps30_Inf2(GTGATGTGTGGTAATAAAAAGCTGAATTCTTAGTCCTCTTCATTCAGCGGGATCGGGTG) (SEQ IDNO: 104).

The PCR products were ligated in the plasmids SaCP816-CPRm-pCWori (SEQID No 74) or SaCP10374-CPRm-pCWOri (SEQ ID NO 82) digested with theHindIII restriction enzyme and using the In-Fusion Dry-Down PCR CloningKit (Clontech, Takara Bio Europe), providing the new plasmidsSaCP816-CPRm-SaTPS647-pCWori (SEQ ID NO 83),SaCP10374-CPRm-SaTPS647-pCWori (SEQ ID NO 84),SaCP816-CPRm-SaTPS30-pCWori (SEQ ID NO 85),SaCP10374-CPRm-SaTPS30-pCWori (SEQ ID NO 86), SaCP816-CPRm-AaBFS-pCWori(SEQ ID NO 87), SaCP10374-CPRm-AaBFS-pCWori (SEQ ID NO 88),SaCP816-CPRm-PaAFS-pCWori (SEQ ID NO 89), SaCP10374-CPRm-PaAFS-pCWori(SEQ ID NO 90), SaCP10374-CPRm-ClTps2-pCWori (SEQ ID NO 91), andSaCP10374-CPRm-SaTps8201-pCWori (SEQ ID NO 92).

The in-vivo production of oxygenated sesquiterpenes in E. coli cellsusing the above plasmids was performed as described in example 17. Allrecombinant bacteria cells transformed with these plasmids produced theexpected sesquiterpene hydrocarbons as well as the correspondingoxygenated products also observed in the in-vitro experiments (FIGS. 22to 26).

What is claimed is:
 1. A method of producing an sesquiterpene alcoholcomprising : i) contacting a terpene of Formula I:

with a polypeptide comprising an amino acid sequence having at least 90%sequence identify to a polypeptide selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 79, and SEQ ID NO: 81; and ii) optionally isolating thealcohol, wherein R is a saturated, mono-unsaturated or poly-unsaturatedaliphatic group composed of 9 carbons and wherein R can be a branchedchain or composed of one or more non-aromatic rings.
 2. The method ofclaim 1, wherein the alcohol comprises α-sinensol, β-sinensol,α-santalol, β-santalol, α-trans-bergamotol, epi-β-santalol, lanceloland/or mixtures thereof.
 3. A method of producing a sesquiterpenealcohol comprising α-sinensol, β-sinensol, α-santalol, β-santalol,α-trans-bergamotol,epi-β-santalol, lancelol and/or mixtures thereofcomprising contacting α-farnesene, β-farnesene, α-santalene,β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolenewith a polypeptide having a P450 monoxygenase activity wherein thesesquiterpene alcohol produced comprises at least about 36% of a cisisomer.
 4. The method of claim 3, wherein the sesquiterpene alcoholproduced comprises at least 46%, 50%, 72%, 96% or 100% of a cis isomer.5. The method of claim 3, wherein the polypeptide comprises an aminoacid sequence having at least 90%, 95%, 98%, or 100% sequence identityto a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 71, and SEQ ID NO:
 73. 6. The method of claim 4, wherein thepolypeptide comprises an amino acid sequence having at least 90%, 95%,98% or 100% sequence identity to a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO:
 73. 7. An isolatedpolypeptide having monooxygenase activity comprising an amino acidsequence having at least 90%, 95%, 98% or 100% sequence identity to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 79, and SEQID NO:
 81. 8. An isolated nucleic acid molecule comprising: i) thenucleic acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:78 or SEQ ID NO: 80; or ii) a nucleic acid molecule that encodes thepolypeptide of claim
 7. 9. A method for producing a polypeptide havingP450 monoxygenase activity comprising transforming a host cell ornon-human organism with the nucleic acid of claim 8; and culturing thehost cell or organism under conditions that allow for the production ofthe polypeptide.
 10. The method of claim 3 comprising i) cultivating anisolated cell under conditions suitable to produce a P450 polypeptidehaving monooxygenase activity, wherein the cell: a) produces a acyclicpyrophosphate terpene precursor; b) expresses a P450 reductase, c)expresses a polypeptide that has α-farnesene, β-farnesene, α-santalene,β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolenesynthase activity and that produces one ore more α-farnesene,β-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene, and d) expresses the polypeptidehaving P450 monooxygenase activity; and ii) optionally isolating thealcohol from the cell.
 11. The method of claim 10, wherein the acyclicpyrophosphate terpene precursor is selected from the group consisting ofgeranyl-pyrophosphate (GPP), farnesyl-diphosphate (FPP) andgeranylgeranyl-pyrophosphate (GGPP).
 12. A vector comprising i) thenucleic acid molecule of claim 8; or ii) a nucleic acid encoding apolypeptide having a P450 monoxygenase activity comprising an amino acidsequence having at least 90% sequence identity to SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 78 orSEQ ID NO:
 80. 13. The vector of claim 12, wherein the vector is aprokaryotic vector, viral vector or a eukaryotic vector.
 14. The vectorof claim 12, wherein the vector is an expression vector.
 15. A host cellor non-human organism comprising the nucleic acid molecule of claim 8,or a vector comprising said nucleic acid molecule.
 16. The method ofclaim 10, wherein the cell is a prokaryotic cell or a eukaryotic cell.17. The method of claim 16, wherein the prokaryotic cell is a bacterialcell.
 18. The method of claim 16, wherein the eukaryotic cell is a yeastcell or a plant cell.
 19. The method of claim 1 comprising i)cultivating an isolated cell under conditions suitable to produce thepolypeptide having P450 monooxygenase activity, wherein the cell: a)produces a acyclic pyrophosphate terpene precursor; b) expresses a P450reductase, c) expresses a polypeptide that has α-farnesene, β-farnesene,α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/orβ-bisabolene synthase activity and that produces one or moreα-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene,epi-β-santalene, and/or β-bisabolene, and d) expresses the polypeptide;and ii) optionally isolating the alcohol from the cell.
 20. The methodof claim 1, wherein step a) comprises cultivating a non-human hostorganism or cell capable of producing a acyclic pyrophosphate terpeneprecursor and transformed to express one or more of the polypeptide.